Human immunodeficiency virus type 1 requires interactions with CD4 and coreceptors for infection. The chemokine receptors, CCR5 and CXCR4 are major coreceptors and all HIV-1 isolates use one or both. CCR5-using (R5) viruses account for almost all transmissions. Preliminary data presented shows that R5 viruses vary considerably in cell tropism depending on their capacity to exploit low levels of CD4 and/or CCR5 for infection. Thus, R5 tropisms can be described as narrow/R5 and broad/R5. Broad/R5 envelopes conferred infection of macrophages and T-cells that expressed low levels of CD4 and/or CCR5, while narrow/R5 envelopes only infected cells with high CD4 levels. Preliminary data also suggests that narrow/R5 envelopes are more resistant to neutralizing antibodies. Of note, envelopes with broad/R5 tropism were identified in immunoprivileged brain tissue. Our hypothesis is that neutralization sensitive, broad/R5 viruses evolve late in disease when immunity declines or in immunoprivileged tissues. Such viruses may preferentially transmit and predominate in acute infection before neutralizing antibodies arise. The origins and phenotypes of HIV-1 in semen are poorly defined. Whether R5 strains that confer broad tropism are present is not known. Several distinct sources may contribute to the pool of virus in semen, including both immune and immunoprivileged tissues e.g. the testes. For some individuals, HIV-1 sequences in semen show distinct lineages from those in blood. The extent HIV-1 sequence variation in semen translates into different biological functions e.g. tropism, that affect capacity of HIV-1 to transmit is unknown. Moreover, several studies of donor/recipient transmission pairs demonstrate that transmission is highly selective where only a small subset of viral genotypes are transmitted. This proposal aims to investigate the biological properties of HIV-1 envelopes present in semen and define the characteristics that facilitate transmission into cervical explant cultures. Importantly, will envelopes that confer transmission be vulnerable to neutralizing antibodies? The following three aims are proposed: Aim 1. Analysis of cell tropism and receptor requirements of HIV-1 R5 envelopes amplified from semen and blood sampled at different disease stages. Aim 2. Evaluation of the biological properties of semen and blood derived HIV-1 envelopes including sensitivity to neutralizing antibodies, receptor ligands and envelope interactions with CD4 and CCR5. Aim 3. Assessment of semen envelopes for their capacity to confer infection of cervical explant cultures.